Studies have been performed to examine the molecular basis of positive and negative selection in the thymus, and to determine how TCR heterodimers recognize their ligands. Transgene (tg)-bearing thymocytes from mice that are class I MHC deficient because their beta2 microglobulin (beta2M) genes have been disrupted through homologous recombination (beta2M) failed to signal through their antigen receptors and did not escape the thymus to populate peripheral lymphoid tissue. Flow cytometry analysis revealed that the tg+beta2M- thymocytes failed to express several cell surface markers associated with T-cell activation. In addition, thymocytes from beta2M- mice failed to express the heat stable antigen (HSA-) phenotype of mature thymocytes whereas HSA- gamma/deltatg+ thymocytes are present in beta2M+ mice. We are also studying the expression and function of the endogenous murine superantigens encoded by the open reading frame (ORF) of the LTR of mouse mammary tumor viruses (MMTV). These antigens, known as Mls, cause TCR Vbeta-specific deletions of T cells during intrathymic development. DNAs corresponding to the mRNAs encoding the ORF proteins have been cloned and sequenced. The expression of these ORF gene products has also been examined in various lymphoid cell subsets as well as thymic stromal cell lines, in order to determine the cellular basis for the induction of both central and peripheral tolerance by these superantigens. We have studied the structural basis of antigen recognition by the TCR heterodimer. We have found that an allelic polymorphism constituting a single amino acid substitution in the first complementarity determining region (CDR1) of the murine V~3 protein profoundly influences TCR usage in the MHC-restricted T-cell response to the antigen pigeon cytochrome c (cyt).